Aliidiomarina halalkaliphila sp. nov., a haloalkaliphilic bacterium isolated from a soda lake in Inner Mongolia Autonomous Region, China

A haloalkaliphilic strain (IM 1326T) was isolated from brine sampled at a soda lake in the Inner Mongolia Autonomous Region, China. Cells of the strain were rod-shaped and motile. Strain IM 1326T was able to grow at 4–42 °C (optimum, 37 °C) with 0–13.0 % (w/v) NaCl concentrations (optimum at 4.0–6.0 %) and at pH 7.5–11.0 (optimum at 9.0–10.0). The 16S rRNA gene phylogenetic analysis revealed that the isolate belongs to the genus Aliidiomarina and is closely related to the type strains of Aliidiomarina sanyensis (95.8 % sequence similarity), Aliidiomarina shirensis (95.7 %), Aliidiomarina iranensis (95.4 %) and Aliidiomarina haloalkalitolerans (95.3 %). The whole genome of strain IM 1326T was sequenced, and the genomic DNA G+C content was 49.7 mol%. Average nucleotide identity, average amino acid identity and digital DNA–DNA hybridization values between the isolate and the related Aliidiomarina species were 68.1–84.9 %, 76–78 % and 18.4–20.4 %, respectively. The respiratory quinone was ubiquinone-8. The polar lipid profile included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminophospholipid. The predominant cellular fatty acids were summed feature 9 (10-methyl-C16 : 0/iso-C17 : 1 ω9c, 22.2 %), iso-C15 : 0 (16.1 %) and iso-C17 : 0 (13.1 %). Based on the results of phylogenetic analysis, genome relatedness, and the physiological and chemotaxonomic properties of the isolate, strain IM 1326T is considered to represent a novel species of the genus Aliidiomarina , for which the name Aliidiomarina halalkaliphila sp. nov. is proposed (type strain IM 1326T=CGMCC 1.17056T=JCM 34227T).


INTRODUCTION
The genus Aliidiomarina belongs to the family Idiomarinaceae, order Alteromonadales, class Gammaproteobacteria [1]. This genus has two sister genera named Idiomarina and Pseudidiomarina in the List of Prokaryotic names with Standing in Nomenclature. Despite the reclassification of the genus Pseudidiomarina into the genus Idiomarina, the species of both genera could not be distinguished from each other using phenotypic or chemotaxonomic characteristics [2], and recently the genus Pseudidiomarina was reinstated following the results of genome-based analysis [3]. The genus Aliidiomarina was close to the genus Pseudidiomarina in the phylogenetic tree based on the 16S rRNA genes. However, it is distant from both sister genera in the phylogenomic tree based on the 78 core genes [3]. At the time of writing, a total of ten species of this genus were validly published, including Aliidiomarina taiwanensis [1], Aliidiomarina haloalkalitolerans [4], Aliidiomarina shirensis [5], Aliidiomarina minuta [6], Aliidiomarina iranensis [7], Aliidiomarina sanyensis [8], Aliidiomarina soli [9], Aliidiomarina maris [10], Aliidiomarina celeris [11] and Aliidiomarina sedimenti [12]. These species were isolated from a wide variety of habitats, including seawater [1,4], saline lakes [5,6], coastal-marine wetland [7], Spirulina platensis cultivation pool [8], soil [9] and sediments [10][11][12]. Members of the genus Aliidiomarina are Gram-stain-negative, rod-shaped, aerobic and require sodium ions for growth. The major isoprenoid quinone OPEN ACCESS is ubiquinone-8, and iso-branched fatty acids including iso-C 17 : 0 , summed feature 9 (comprising iso-C 17 : 1 ω9c and/or 10-methyl C 16 : 0 ) and iso-C 15 : 0 are the major cellular fatty acid components [1]. This article describes a haloalkaliphilic strain isolated from a soda lake. The strain is proposed as a novel species of the genus Aliidiomarina.

ISOLATION AND MAINTENANCE
Strain IM 1326 T was isolated from a brine sample of a soda lake (Hutong Qagan Lake) from the Ordos, Inner Mongolia Autonomous Region of China, in July 2017. The sampling site was located at the position of 39° 12′ 14″ N, 109° 0′ 8″ E, and an altitude of 1250 m. The total salinity of the lake water was 26%, while the pH was 9.95. The brine sample was stored in a sterilized plastic bottle and transported to the laboratory at ambient temperature. The sample was incubated at 37 °C for 2 weeks after being inoculated in LN liquid medium (originally designed for microbe using light by refering neutral oligotrophic medium) composed of (per litre): 15.0 g NaCl, 0.5 g yeast extract (Difco), 6.0 g NaHCO 3 , 4.0 g Na 2 CO 3 , 0.2 g NH 4 Cl, 2.0 g KCl, 0.25 g fish peptone, 0.38 g sodium formate, 0.25 g sodium acetate, 0.25 g sodium pyruvate, 1.0 g MgSO 4 ·7H 2 O, 0.08 g CaCl 2 , 56 mg FeSO 4 ·7H 2 O, and pH was adjusted to 9.5 with 1 M NaOH. The enrichment culture was spread on agar plates of the same medium (15.0 g agar per litre) and incubated at 37 °C for 5 days. Pure cultures were obtained by repeated subculturing of a single colony on the same solid medium. A circular, convex and light brown colony was obtained on LN plates after 5 days of incubation at 37 °C. The strain was routinely cultured at 37 °C in LN medium. Stock culture of the isolate was preserved in LN medium with 20 % glycerol (v/v) at −80 °C.

PHYLOGENETIC ANALYSIS BASED ON 16S rRNA GENE SEQUENCES
The phylogenetic position of strain IM 1326 T was determined through 16S rRNA gene sequence analysis. The 16S rRNA gene sequence was amplified using oligonucleotide primers complementary to highly conserved regions of bacterial 16S rRNA genes. The forward and reverse primers were 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), respectively [13]. The amplified product was cloned in the pMD-18T vector (Takara), and the recombinant plasmids were transformed into Escherichia coli JM109 (BioMed) cells. Sanger sequencing was performed by Witon Information Technology Co., Ltd. (Beijing, PR China). Sequences were preliminarily analysed by the blast program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the EzBioCloud webserver (www.ezbiocloud.net), and the closely related sequences were selected as references from the list of hits with valid names. The maximum-likelihood, neighbour-joining, minimum-evolution and maximum-parsimony methods were used to reconstruct the phylogenetic trees in mega 6.0 software [14]. The robustness of the phylogenetic tree topology was evaluated by bootstrap analyses based on 1000 replicates [15].  (Fig. 1). Considering the low bootstrap values (not shown if less than 70%), the topology of the phylogenetic tree was not stable. The topology and phylogenetic relationship were also supported by the neighbour-joining, minimum-evolution and maximum-parsimony phylogenetic trees (Figs S1-S3, available in the online version of this article). In addition, the genera Pseudidiomarina and Idiomarina were closer in most phylogenetic trees (Figs 1, S1 and S2), apart from the maximum-parsimony tree (Fig. S3). Moreover, the bootstrap values were lower than 70 % between the genus Aliidiomarina and the clade of the genera Pseudidiomarina and Idiomarina. This finding is different from the previous report using FastTree with JTT+CAT parameters [3]. Altogether, strain IM 1326 T was likely to represent a novel species of the genus Aliidiomarina.

GENOME FEATURES AND PHYLOGENOMIC ANALYSIS
The draft genome of strain IM 1326 T was sequenced by Biomarker Technologies (Beijing, PR China) for genome-level phylogenetic analysis. Briefly, the genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method, and purity was ascertained using a Qubit 3.0 fluorometer (Invitrogen). After the construction of the DNA library, the genome was sequenced using an Illumina NovaSeq platform. Genome assembly was carried out using SPAdes version 3.13.0. The draft genome sequence has been deposited with accession number VJWL00000000 in the GenBank of the NCBI nucleotide database (www.ncbi.nlm. nih.gov/genome) and annotated using the NCBI Prokaryotic Genome Annotation Pipeline version 4.7 (www.ncbi.nlm.nih.gov/ genome/annotation_prok/). DNA G+C content and other detailed information were obtained from the genome sequence. The average nucleotide identity (ANI) percentages with other species of the genus Aliidiomarina were calculated using the blast (ANIb) and MUMmer (ANIm) algorithms from the JSpecies Web Server (http://jspecies.ribohost.com/jspeciesws) [16]. The average amino acid identity (AAI) values were calculated using the online tool ANI/AAI-Matrix (http://enve-omics.ce.gatech.edu/ g-matrix/), a genome-based distance matrix calculator. In addition, genome-to-genome distance analysis via digital DNA-DNA hybridization (dDDH) was performed with the Genome-to-Genome Distance Calculator (http://ggdc.dsmz.de/distcalc2.php) using the method described by Meier-Kolthoff et al. [17]. Furthermore, a phylogenomic tree was reconstructed based on 120 bacterial conserved single-copy genes obtained by employing the Genome Taxonomy Database [18].
Genome sequencing of strain IM 1326 T resulted in raw data of 3.06 Gb with 1189× genome coverage. Sequence reads were de novo assembled into 19 contigs and 12 scaffolds. The genomic size was 2.57 Mb. The genomic DNA G+C content of strain IM 1326 T was estimated to be 49.7 mol%, which was similar to that of other species of the genus Aliidiomarina (46.3-52.1 mol%) and slightly higher than that of A. taiwanensis AIT1 T (48.7 mol%), the type species of the genus Aliidiomarina (Table 1). The detailed genomic information is presented in Table S1. The value of genome relatedness between strain IM 1326 T and its closely related species ranged from 68.1 to 72.6 % for ANIb, 80.7 to 84.9 % for ANIm, 76 to 78 % for AAI, and 16.2 to 22.7 % for dDDH ( Table 2). The percentage similarity values with A. sanyensis GYP-17 T , the most closely related species, were 72.6 % for ANIb, 83.5 % for  celeris F3105 T [9], and A. sedimenti GBSy1 T [10].  The maximum-likelihood phylogenomic tree reconstruction based on 120 bacterial conserved single-copy genes revealed that strain IM 1326 T was located in the Aliidiomarina clade and separated from other species (Fig. 2). The bootstrap value between strain IM 1326 T and A. sanyensis GYP-17 T was 100%, suggesting it was a novel species of Aliidiomarina. The neighbour-joining, minimum-evolution and maximum-parsimony phylogenomic trees displayed similar phylogenetic positions (Figs S4-S6). In addition, the phylogenomic trees based on 120 bacterial conserved single-copy proteins agreed with the tree based on the 78 concatenated core protein sequences of the family Idiomarinaceae [3]. Therefore, the results confirmed that IM 1326 T represents a novel species in the genus Aliidiomarina. Tests for catalase and oxidase activities and the hydrolysis of starch, gelatin, casein, and Tweens 20, 40, 60, and 80 were carried out using routine cultivation methods on HM medium with 4 % (w/v) NaCl as described by Harrigan et al. [21,22]. H 2 S formation was detected using a filter-paper strip impregnated with lead acetate [23]. Indole production from tryptophan, the utilization of sugars and organic acids, and the Voges-Proskauer test were assessed as described by Oren et al. [24]. In brief, the utilization of sole carbon and energy sources was detected using minimal medium (tryptone was omitted and yeast extract was   Table 2. Continued decreased to 1 g l −1 ) with various supplementary carbon sources and then comparing the cell density. Acid production from sugars and alcohols was determined, and the substrates used were d-glucose, d-galactose, sucrose, d-sorbitol, mannose, maltose, starch, d-fructose, l-sorbose, ribose, trehalose, d-xylose and glycerol. Production of acid from the above-mentioned carbohydrates and sugar alcohols was tested on the growth medium supplemented with 10.0 g l −1 of the substrate. The pH of the cultures was measured with a glass electrode at the end of the incubation period and compared with the initial pH and the pH of the control tube without substrate. Reduction of nitrate and nitrite were detected by using sulfanilic acid and α-naphthylamine reagent [25]. All tests were performed in triplicate along with the reference species under identical conditions.
Strain IM 1326 T was Gram-stain-negative. The cells were approximately 0.3-0.6 µm wide and 0.8-2.1 µm long (Fig. S7). The isolate can move, but no flagellum was observed under the transmission electron microscope (Fig. S8). Furthermore, the type of motility was explored. There were no rafts of cells and isolated cells were observed in the microscope image of the edge of colonies (Fig. S9), indicating that the cells did not move by gliding; therefore, further exploration is necessary. The colonies were light brown when grown on HM medium (pH 8.5) for 24 h at 37 °C. Strain IM 1326 T was able to grow in presence of 0-13.0 % NaCl concentration (optimum, 4.0-6.0 %), at 4-42 °C (optimum, 37 °C) and pH 7.5-11.0 (optimum, pH 9.0-10.0). Moreover, it showed more tolerance to alkalinity (up to pH 11.0) than most of the reference species. Strain IM 1326 T could hydrolyse gelatin, casein and different kinds of Tweens, e.g. Tween 20, 40 and 60, but could not hydrolyse starch. In addition, strain IM 1326 T was positive for nitrate reduction, but negative for nitrite reduction. Cells of strain IM 1326 T were oxidase-and catalase-positive. Correspondingly, a respiratory nitrate reductase operon (including four genes FM042_RS01865 to FM042_RS01880), NarK family nitrate/nitrite transporter (FM042_RS01935) and catalase (FM042_RS03320 and FM042_RS09450) were annotated in the genome. However, the draft genome did not contain the gene for amylase and nitrite reduction. Strain IM 1326 T could produce H 2 S from cysteine but did not produce indole from tryptophan. The detailed morphological, physiological and biochemical characteristics of strain IM 1326 T are provided in the species description and Table 1.  Table S2. The predominant fatty acids detected in strain IM 1326 T were 10-methyl-C 16 : 0 /iso-C 17 : 1 ω9c (summed feature 9, 22.2 %), iso-C 15 : 0 (16.1 %) and iso-C 17 : 0 (13.1 %). These are also the dominant fatty acids in the two reference strains. In addition, we observed that, contrary to the original report, the content of C 16 : 0 was high (up to 13.2 %) in A. taiwanensis CGMCC 1.15255 T [1]. However, the amount of C 16 : 0 was lower in IM 1326 T and A. haloalkalitolerans DSM24939 T (Table S2). These data on the fatty acids composition of two reference species were similar to the previous report [2]. Despite the divergence in results from the different labs, all three species contained the major fatty acids of the genus Aliidiomarina [1].
Polar lipids of strain IM 1326 T were extracted by a phenol-chloroform system and analysed by using two-dimensional thin-layer chromatography (TLC) as described previously [26]. Merck silica gel 60 F254 aluminum-backed thin-layer plates were used for the TLC analysis. The first and second dimensional TLC solvents were chloroform-methanol-water (65 : 25 : 4, by vol.) and chloroform-methanol-acetate-water (80 : 12 : 15 : 4, by vol.), respectively. The reagent sulfuric acid-ethanol (1 : 1, v/v) was used to detect total polar lipids. According to the TLC chromatogram, the major polar lipids of strain IM 1326 T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminophospholipid (Fig. S10). The polar lipid composition of two reference species (including the type species) were identified under identical experimental condition (Fig. S10). Most of the Aliidiomarina species contained diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine ( Table 1). The above findings are in unison with the polar lipid compositions of the genus Aliidiomarina [1].
Respiratory quinones were extracted from lyophilized cells, and the extracts were purified by TLC and analysed by highperformance liquid chromatography according to the method of Collins [27]. Strain IM 1326 T contained ubiquinone-8 as the sole respiratory quinone (Table 1). However, a minor quinone of the genus Aliidiomarina ubiquinone-9 was not detected [1].
Consequently, on the basis of the phylogenetic, physiological and chemotaxonomic data, strain IM 1326 T is considered to represent a novel species of the genus Aliidiomarina, for which the name Aliidiomarina halalkaliphila sp. nov. is proposed. Cells are Gram-stain-negative, motile, rod-shaped, approximately 0.3-0.6 µm wide and 0.8-2.1 µm long. Colonies grown for 24 h at 37 °C on HM medium are circular, smooth, convex, light brown and approximately 1.0-3.0 mm in diameter. Oxidase and catalase are positive. Growth occurs at 4-45 °C (optimum, 37 °C), at pH 7.5-11.0 (optimum, pH 9.0-10.0) and with 0-13.0 % (w/v) NaCl concentration (optimum 4.0-6.0 %). Anaerobic and microaerobic growth does not occur on HM medium with or without nitrate. Nitrate is reduced, but nitrite is not. H 2 S is produced from cysteine. Production of indole and utilization of urea does not occur. The methyl red reaction is negative, but the Voges-Proskauer test result is positive. Gelatin, casein, DNA and Tweens 20, 40 and 60 are hydrolysed, but Tween 80, aesculin and starch are not hydrolysed. The following substrates are utilized as single carbon and energy sources for growth: l-arginine, l-lactate, l-glutamate, acetate, l-malate, pyruvate, succinate, citrate, l-serine, glycine, valine and histidine. The following substrates are not utilized as sole sources of carbon and energy: d-glucose, d-galactose, sucrose, d-sorbitol, fumarate, l-aspartate, mannose, maltose, starch, l-alanine, l-ornithine, d-fructose, l-sorbose, ribose, trehalose, d-xylose, glycerol and l-lysine. No acids are produced with the following substrates: d-glucose, d-galactose, sucrose, d-sorbitol, mannose, maltose, starch, d-fructose, l-sorbose, ribose, trehalose, d-xylose and glycerol. The respiratory quinone is ubiquinone-8. The major cellular fatty acids of the isolate are 10-methyl-C 16 : 0 /iso-C 17 : 1 ω9c (summed feature 9), iso-C 15 : 0 and iso-C 17 : 0 . The major polar lipids include diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminophospholipid.

DESCRIPTION OF
The type strain, IM 1326 T (=CGMCC 1.17056 T =JCM 34227 T ), was isolated from the soda lake Hutong Qagan Lake in Inner Mongolia Autonomous Region, China. The genomic size of the type strain is 2.57 Mb, and DNA G+C content is 49.7 mol% (determined from the genome sequence).

Funding information
This study was supported by the National Natural Science Foundation of China (No. 91751201) and the Science and Technology Basic Resources Investigation Project (No. 2017FY100300).

Conflicts of interest
The authors declare that there are no conflicts of interest.